Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease resulting in paralysis and death usually within 3-5 years of diagnosis1. The landmark discovery in 1993 of pathogenic mutations of Cu/Zn superoxide dismutase (SOD1) in a subset of familial ALS has spurred research into the mechanism of SOD1-related ALS (SOD1-ALS)2. A combination of genetic and biophysical techniques has shown that SOD1 point-mutations produce a toxic gain-of-function, the exact nature of which remains unresolved1. One proposed mechanism is that misfolding and aggregation of the mutant SOD1 protein underlies toxicity. Inclusion bodies immunoreactive for SOD1 are present in human cases of SOD1-ALS and in transgenic ALS models3. The mechanism(s) by which in vivo misfolded and aggregated SOD1 exhibits toxicity have been proposed to involve overwhelming the protein-folding chaperone system4,5, inhibition of proteasomes6, or aberrant interactions with mitochondrial proteins such as Tom207 or Bcl-28. Another proposed gain-of-function for mutant SOD1 is reduced zinc binding9, resulting in the transformation of the protein into a toxic pro-oxidant10. It is hypothesized that the root of the toxic gain-of-function is an alteration in the structure of SOD1, one of the most stable cytoplasmic proteins11. This may be either through exposure of novel sites of interaction in SOD1 or loss of structure in the metal binding loops, but this has not been demonstrated in vivo. The in vivo demonstration that SOD1 misfolding is linked to ALS pathogenesis is required to substantiate the SOD1-ALS hypotheses and yield insight into the mechanism of SOD1-ALS.
The inventors created an in vitro model system12 to study the in vivo SOD-1 misfolding pathway. The inventors hypothesized that the seemingly disparate theories of aberrant pro-oxidant activity and misfolding in SOD1-ALS are in fact linked: SOD1's normal antioxidant role incurs an occupational hazard of being oxidized and this is further exacerbated by its long half-life in motor neurons12. The accumulation of such oxidative insults could promote misfolding and aggregation. SOD1 normally exists as an obligate homodimer, each subunit of which binds one copper and one zinc atom. The inventors have previously shown that mutant SOD1 is more prone to oxidation-induced misfolding than wild-type SOD1 in vitro, and that both form unnatural partially folded monomeric and soluble oligomeric intermediates prior to aggregation13. There is a need to better understand the underlying mechanism of this disease and to determine if SOD1-ALS pathogenesis results from protein misfolding.